Tumor-specific P450 protein

ABSTRACT

The discovery that Cyp1B1 protein is detectable in a wide range of human cancers of different histogenetic types, but is not detectable in non-cancerous tissues, gives rise to diagnostic methods for detecting tumors based on this protein as a marker, and to the possibility of tumor therapies involving the protein. A diagnostic method may include the steps of: (a) obtaining from a patient a tissue sample to be tested for the presence of cancer cells; (b) producing a prepared sample in a sample preparation process; (c) contacting the prepared sample with an antibody that reacts with human Cyp1B1 protein; and (d) detecting binding of the antibody to CYP1B1 protein in the prepared sample.

Work on this invention was supported in part by NIH Grant No. ES-07009.Therefore, the U.S. government may have certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to tumour diagnosis and therapy, and to materialsand methods for use therein.

More particularly, the invention is based on the identification of acytochrome P450 form, specifically CYP1B1, in a wide range of tumours,with a high frequency of expression in each type, and proposes the useof this enzyme as a tumour marker, and as the basis of a selectivetherapeutic approach involving the design of drugs, eg which areactivated to a cytotoxic form by the action of CYP1B1.

BACKGROUND TO THE INVENTION

The major goal of cancer chemotherapy is the development of anti-cancerdrugs that are effective in a wide range of cancers and produce no toxiceffects in normal tissues. The target of such drugs should be expressedonly in tumour cells and not normal cells. However, to date, no suchtumour specific target, general to all types of cancers, has beenidentified.

The cytochromes P450 are a multi-gene family of constitutive andinducible enzymes, which have a central role in the oxidative metabolicactivation and detoxification of both a wide range of xenobiotics (2-4)and several groups of endogenous compounds active in cell regulation andcell signalling including arachidonic acid (5), steroid hormones (6) andfatty acids (7). The major families of P450 involved in xenobioticmetabolism each consist of several individual forms with differentregulatory mechanisms and substrate specificities (2). The majority ofP450s are primarily expressed in liver (2) although individual P450forms are also expressed in specific extra-hepatic tissues (8) includingsmall intestine, kidney and lung.

The human CYP1 gene family (individual P450 forms are identified by theprefix CYP in accordance with the current P450 nomenclature (3)), whichis one of the major P450 families involved in the metabolism ofxenobiotics, is now known to consist of three individual formsclassified into two sub-families. The CYP1A subfamily contains twohighly homologous and well characterised but distinct members. CYP1A1(9) and CYP1A2 (10). CYP1A1 is an inducible P450 expressed primarily inextraheptic tissues (11) while CYP1A2 is a major form of P450 that isconstitutively expressed in liver (12). Recently a second human CYP1subfamily has been identified which to date contains one member, CYP1B1(1). This P450 is dioxin-inducible, and sequence analysis of CYP1B1shows 40% homology with both CYP1A1 and CYP1A2. Although CYP1B1 isassigned to the CYP1 family on the basis of its sequence, it appears tobe structurally distinct from both CYP1A1 and CYP1A2.

Several forms of P450 are considered to have an important role in tumourdevelopment since they can metabolise many potential carcinogens andmutagens (13). Moreover, P450 activity may influence the response ofestablished tumours to anti-cancer drugs; several cancerchemotherapeutic agents can be either activated or detoxified by thisenzymes system (14). The presence of individual forms of P450 hadpreviously been investigated in different types of cancer includingbreast cancer (15), lung cancer (16), colon cancer (17) and head andneck cancer (18) to determine if intra-tumour metabolism of anti-canceragents by P450 could occur and thus influence the response of tumours tothese agents. These studies have generally shown that the level of theP450 forms investigated is significantly reduced or absent in tumourswhen compared with the adjacent normal tissue in which the tumours havedeveloped. However, our recent studies of several different types ofcancer (19) including breast cancer, oesophageal cancer and soft tissuesarcomas have shown that there may be tumour-specific expression of aCYP1 form of P450.

Although CYP1B1 mRNA had previously been identified by Northern blottingin several normal human tissues (1), the presence CYP1B1 protein itselfhad not been demonstrated.

SUMMARY OF THE INVENTION

The present invention is based on the discovery that CYP1B1 is atumour-specific form of P450, present in a wide range of malignanttumours and not detected in normal tissues.

Accordingly, a first aspect of the present invention provides a methodfor the identification of tumour cells, which method comprises the useof a recognition agent, for example an antibody, recognising CYP1B1protein to contact a sample of tissues, cells, blood or body product, orsamples derived therefrom, and screening for a positive response. Thepositive response may for example be indicated by an agglutinationreaction or by a visualisable change such as a colour change orfluorescence, eg immunostaining, or by a quantitative method such as inuse of radio-immunological methods or enzyme-linked antibody methods.

The method therefore typically includes the steps of (a) obtaining froma patient a tissue sample to be tested for the presence of cancer cells;(b) producing a prepared sample in a sample preparation process; (c)contacting the prepared sample with a recognition agent, such as anantibody, that reacts with human CYP1B1 protein; and (d) detectingbinding of the recognition agent to CYP1B1 protein, if present, in theprepared sample. The human tissue sample can be from for example thebladder, brain, breast, colon, connective tissue, kidney, lung, lymphnode, oesophagus, ovary, skin, stomach, testis, and uterus.

A preferred sample preparation process includes tissue fixation andproduction of a thin section. The thin section can then be subjected toimmunohistochemical analysis to detect binding of the recognition agentto CYP1B1 protein. Preferably, the immunohistochemical analysis includesa conjugated enzyme labelling technique. A preferred thin sectionpreparation method includes formalin fixation and wax embedding.Alternative sample preparation processes include tissue homogenization,and preferably, microsome isolation. When sample preparation includestissue homogenization, a preferred method for detecting binding of theantibody of CYP1B1 protein is Western blot analysis. Alternatively, animmunoassay can be used to detect binding of the antibody to CYP1B1protein. Examples of immunoassays are antibody capture assays,two-antibody sandwich assays, and antigen capture assays. Preferably,the immunoassays is a solid support-based immunoassay. When Western blotanalysis or an immunoassay is used, preferably it includes a conjugatedenzyme labelling technique.

Although the recognition agent will conveniently be an antibody, otherrecognition agents are known or may become available, and can be used inthe present invention. For example, antigen binding domain fragments ofantibodies, such as Fab fragments, can be used. Also, so-called RNAaptomers may be used (36, 37). Therefore, unless the contextspecifically indicates otherwise, the term “antibody” as used herein isintended to include other recognition agents. Where antibodies are used,they may be polyclonal or monoclonal. Optionally, the antibody canproduce by a method so that it recognizes a preselected epitope of saidCYP1B1 protein.

A second aspect of the invention lies in the presence of CYP1B1 proteinselectively in tumours, eg in kidney tumours and not normal renaltissue, combined with the absence of CYP1B1 protein expression in normalliver, which provides a mechanism for the selective targeting ofanti-cancer drugs based on CYP1B1 metabolism in tumours. Drugs can bedesigned for, or screened for, specific metabolism by CYP1B1 in tumourswhereby this metabolism converts a non-toxic moiety into a toxic one,which kills or inhibits the tumour or makes it more susceptible to otheragents.

A third aspect of the invention provides for the targeting of cytotoxicdrugs or other therapeutic agents, or the targeting of imaging agents,by virtue of their recognition of CYP1B1 epitopes on the surface of atumour cell, whether as part of the complete CYP1B1 protein itself or insome degraded form such as in the presentation on the surface of a cellbound to a MHC protein.

Another aspect of the invention provides stimulation of the immunesystem of cancer patients, for example by activating cytotoxic or helperT-cells which recognise CYP1B1 epitopes so as to implement acell-mediated or humoral immune response against the tumour. Theactivation of the immune system can be achieved by immunisation withCYP1B1 sequences.

Because the expression of CYP1B1 is very common in tumours of manydifferent types, it is likely that this enzyme performs an essentialfunction for the tumour cells, for example by inactivating endogenousanti-tumour compounds such as 2-methoxyestradiol. Consequently, anotheraspect of the invention is the reduction of CYP1B1 levels in tumourcells, for example by the use of suicide inhibitors or by usingantisense RNA methods to decrease the synthesis of the protein.Down-regulation of the CYP1B1 promoter could also achieve the reductionof CYP1B1 levels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SDS-PAGE and immunoblotting procedures using an anti-CYP1B1antibody to look for CYP1B1 protein in normal and tumorous kidney tissueand in normal liver tissue.

FIG. 2 shows SDS-PAGE and immunoblotting to detect CYP1B1 protein inbreast tumour and in normal liver tissue.

FIG. 3 shows and immunoblot of CYP1B1 in different types of tumours andnormal tissues.

FIG. 4 shows CYP1B1 and β-actin mRNA in normal (A and B) andcorresponding tumour (C and D) samples which have been detected byRT-PCR.

DETAILED DESCRIPTION OF THE INVENTION

Expression of CYP1B1 was investigated in different types of cancers thathad developed in a broad range of different anatomical sites (bladder,breast, colon, kidney, lung, oesophagus, ovary, skin, stomach, uterus,bone and connective tissue, lymph node, brain and testis). Primarymalignant tumours of these tissues constitute different histogenetictypes (carcinomas, lymphomas, sarcomas, neuro-epithelial tumours andgerm cell tumours) each with a different biological behaviour. Thesetumours also represent a range of both common and less common types ofcancer. The presence of CYP1B1 was also investigated in a wide varietyof normal tissues.

Immunohistochemistry for CYP1B1 showed that in all the different typesof tumour there was strong immunoreactivity for CYP1B1. CYP1B1immunoreactivty was localised specifically to tumour cells. Non-tumourcells including stromal cells, inflammatory cells, and endothelial cellspresent in the sections of tumour showed no immunoreactivity for CYP1B1.

There was no significant intra-tumour heterogeneity of CYP1B1immunoreactivity and only in five out of 133 tumours was CYP1B1 notdetected. There was no immunoreactivity for CYP1B1 in any of the normaltissues studied which included liver, kidney, small intestine and lung.

The absence or low level of individual forms of P450 in most studies ofhuman cancer (15-18), combined with extrapolation from studies of rodenthepatic carcinogenesis (25), had led to the general belief that tumourcells do not significantly express P450. However, we have now shown thatCYP1B1 is expressed in a wide variety of malignant tumours of differenthistogenetic types and is not present in normal tissues, indicating thatthis P450 is a tumour specific form of P450. Tumours are composed of avariable proportion of tumour cells and non-tumour cells. To identifythat a protein is tumour specific, it is important to demonstrate thatthe protein is localised only to tumour cells. Immunohistochemistryallows the direct visualisation of tumour cells and has the spatialresolution to separate tumour cells from non-tumour cells. Furthermore,it is important to show there has been no differential degradation ofproteins in normal tissue samples compared with tumour samples, andimmunoblotting for β-actin (as a positive control protein) showed it tobe present in every normal and tumour sample indicating there was noprotein degradation. In addition, Coomassie blue staining of thepolyacrylamide gels showed no evidence of protein degradation. Moreover,immuno-histochemistry of the tumour samples provides its own internalcontrol as sections of tumour contain non-tumour cells.

The presence of CYP1B1 in many types of tumour suggests that this P450may have a crucial endogenous function in tumour cells and CYP1B1 maycontribute to drug resistance that is observed in many types of tumour.CYP1B1 is also likely to be important in tumour development andprogression. Its identification in a diverse range of cancers ofdifferent histogenetic types and is absence from normal tissues appearsto make CYP1B1 one of the common changes of a gene product in malignancy(26).

A previous investigation (1) found mRNA in normal tissues. In sometumours, increased (2-4×) mRNA was found compared with normal. Thismight be due to increased transcription mediated by hypoxia induciblefactor. This is a novel heterodimeric transcription factor which isinduced by hypoxia, and the stimulus in this case may be the hypoxicmicro-environment that can exist in tumours, and this factor can have asone of its components the Ah receptor nuclear translocator (27).However, regulation of other forms of P450 is complex (2, 28) and theregulation of CYP1B1 in tumours is likely to be complex also, withmultiple mechanisms including transcriptional and post-transcriptionalfactors involved.

The tumour-specific expression of CYP1B1 has important consequences forboth the diagnosis and treatment of cancer. New diagnostic proceduresbased on the presence of CYP1B1 in cancer cells can be developed, whilethe expression of CYP1B1 in tumour cells provides a molecular target forthe development of new anti-cancer drugs that are selectively activatedby CYP1B1 in tumour cells. Since CYP1B1 is found in a wide range oftumours it would be expected that such drugs would be effective intreating many different types of cancer. An important feature is that itwould be anticipated these drugs would not be associated with thesystematic toxicity that limits the use of current anti-cancer drugs asCYP1B1 is not present in normal tissues especially liver, smallintestine and kidney that are the main tissues involved in drugmetabolism. Thus, a major problem to targeting anti-cancer drugs attumours based on their selective activation by P450 has been markedhepatic P450 metabolism of drugs resulting in decreased bioavailabilityand/or undue toxicity. The absence of CYP1B1 protein in liver overcomesthis problem.

As regards tumour diagnosis, numerous methods for using antibodies todetect a specific protein, including CYP1B1, in a biological sample areknown and can be used in the present invention. Any of the variousantibody methods can be used alone in practising the present invention.If desired, two or more methods can be used to complement one another.

A preferred method for use in the present invention isimmunohistochemical analysis. Immunohistochemical analysisadvantageously avoids a dilution effect when relatively few cancer cellsare in the midst of normal cells. An early step in immunohistochemicalanalysis is tissue fixation, which preserves proteins in place withincells. This prevents substantial mixing of proteins from differentcells. As a result, surrounding normal cells do not diminish thedetectability of CYP1B1-containing cancer cells. This is in contrast toassay methods that involve tissue homogenization. Upon tissuehomogenization, CYP1B1 protein from cancer cells is mixed with proteinsfrom any surrounding normal cells present in the tissue sample. Theconcentration of CYP1B1 protein is thus reduced in the prepared sample,and it can fall below detectable limits. Immunohistochemical analysishas at least three other advantages. First, it requires less tissue thanis required by alternative methods such as Western blot analysis orimmunoassay. Second, it provides information on the intracellularlocalization and distribution of immunoreactive material. Third,information on cell morphology can be obtained from the same thinsection used to test for the presence of CYP1B1 protein. Preferably,when immunohistochemical analysis is employed in the practice of thisinvention, several thin sections from each tissue sample are preparedand analysed. This increases the chances of finding small tumours.

Another preferred antibody method for use in the present invention isWestern blot analysis, ie sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), followed by immunoblotting. Samplepreparation for Wester blot analysis includes tissue homogenization, andoptionally isolation of microsomes. Western blot analysis has theadvantage of detecting immunoreactivity on proteins that have beenseparated with high resolution, according to (apparent) molecularweight.

Immunoassays such as antibody capture assays, two-antibody sandwichassays, and antigen capture assays can also be used in the presentinvention. Sample preparation for immunoassays include tissuehomogenization, and optionally isolation of microsomes. Immunoassayshave the advantage of enabling large numbers of samples to be testedrelatively quickly, and they offer quantitative precision.

Principles and practice of immunohistochemistry, Western blot analysis,and immunoassays are well known. One of ordinary skill in the art canselect suitable protocols and carry out immunohistochemical analysis.Western blot analysis, or an immunoassay, in the practice of the presentinvention (30).

Experimental details

1. Preparation of antibodies

As already noted, the diagnostic aspects of the present invention canconveniently use an antibody that recognizes human CYP1B1. Antibodyspecificity for CYP1B1 protein is preferable, but not required.Preferably, any non-CYP1B1 protein recognized by the antibody is readilydistinguished from CYP1B1, eg, according to apparent molecular weight ona Western blot. With selection of an appropriate assay protocol, whichis within ordinary skill in the art, the invention can be practised witha polyclonal antibody or a monoclonal antibody.

A polyclonal antibody or monoclonal antibody suitable for use in thepresent invention can be obtained according to conventional procedures(30). Preparation of antibodies that react with CYP1B1 protein is known(31). Procedures for obtaining antibodies that react with human CYP1B1protein can be carried out using a preparation of non-human CYP1B1protein, eg, murine CYP1B1 protein. A CYP1B1 protein preparationsuitable for eliciting antibodies useful in the present invention can beobtained according to various procedures, including those described(31).

Antibodies useful in the present invention can be obtained by immunizingan animal with a preparation containing intact CYP1B1 protein.Alternatively, useful antibodies can be obtained by immunizing an animalwith a polypeptide or oligopeptide corresponding to one or more epitopeson the CYP1B1 protein.

Preparation

To prepare an antibody according to the latter approach, two 15-merpeptides corresponding to epitopes on the human CYP1B1 protein weresynthesized. Each corresponded to a different putative surface loopregion of the CYP1B1 enzyme. The first peptide (designated 217A)consisted of 14 amino acids, ie, ESLRPGAAPR DMMD (SEQ ID NO:1). Peptide217A represented amino acid positions 312-325 of the deduced amino acidsequence. A carboxy terminal cysteine was included for use in aconjugation reaction. The second peptide (designated 218A) consisted of14 amino acids, ie, EKKAAGDSHG GGAR (SEQ ID NO:2). Peptide 218Arepresented positions 332-345 of the deduced amino acid sequence. Acarboxy terminal cysteine was added for use in a conjugation reaction.Each of these peptides was conjugated directly to KLH.

Male New Zealand rabbits were immunized at several anatomical sitesusing 100 μg of the 217A peptide conjugate or the 218A peptideconjugate. The conjugates were dissolved in 300 μl of PBS mixed with 300μl of Freund's Complete Adjuvant. Three weeks after the initialimmunization, the rabbits were boosted with 50 μg of one of each of theconjugates (contained in 300 μl PBS mixed with 300 μl of Freund'sIncomplete Adjuvant, injected in several sites). One week later (fourweeks after the initial injection), the rabbits were boosted again,using the same protocol. One week after this second boost, the firstserum sample was collected. Rabbits were subsequently boosted, and serumsamples were collected, weekly. Serum samples were screened foranti-CYP1B1 titre and specificity by Western blotting against a humanCYP1B1-maltose binding fusion protein expressed in E. coli, and humanCYP1B1 protein expressed in COS-1 cells.

Anti-CYP1B1 IgG was purified by immunoaffinity chromatography. Thechromatography was carried out using the appropriate CYP1B1 peptidelinked directly to a commercial N-hydroxysuccinamide ester of aderivatized, cross-linked agarose gel bead support (AffiGel 10; Biorad,Richmond, Calif.). Conjugation and chromotography were performedaccording to the vendor's recommended protocols.

2. Detection of CYP1B1 protein and its mRNA

In general in the experiments that follow, samples of normal tissue wereobtained from tissue specimens that were removed from patientsundergoing surgery for malignant disease. Normal liver, stomach andsmall intestine were also obtained from organ transplant donors. All thetissue samples were processed immediately after excision to prevent anydegradation of protein or mRNA and ensure no deterioration in tissuemorphology. We have previously shown that human liver obtained in thisway shows no loss or degradation of individual forms of hepatic P450(20). Tissue blocks for immuno-histochemistry were fixed in 10% neutralbuffered formalin for 24 hours and then embedded in wax, while tissuesamples for immunoblotting and mRNA analyses were rapidly frozen inliquid nitrogen and stored at −80° C. prior to use.

The presence of CYP1B1 protein in tissue samples was investigated usingimmunohistochemistry (21) and sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE) combined with immunoblotting (22).Immunohistochemistry ensures the identification of specific types ofcells containing CYP1B1 and is an idea technique for investigating thepresence of CYP1B1 in tumour cells as tumours are composed of a variableproportion of tumour cells and non-tumour cells.

Immunohistochemistry was used to determine the cellular localisation anddistribution of CYP1B1 and was performed on formalin fixed wax embeddedsections using two antibodies which recognise CYP1B1. Sites ofimmunoreactivity were detected using an alkaline phosphataseanti-alkaline phosphatase (APAAP) technique (21). Samples of tumour andnormal tissue were fixed in 10% neutral buffered formalin for 24 hourand then embedded in wax. Sections were cut on to glass slides and forimmunohistochemistry sections of tumours and normal tissues were dewaxedin xylene, rehydrated in alcohol and then washed sequentially in coldwater and 0.05M Tris-HCl (pH 7.6) containing 0.15M sodium chloride(TBS). The sections were then immunostained with the CYP1B1 antibodies.Subsequently, monoclonal mouse anti-rabbit immunoglobulin (1/100, DakoLtd, High Wycombe, Bucks; UK), rabbit anti-mouse immunoglobulin (1/100,Dako) and mouse monoclonal APAAP (1/100, Dako) were sequentially appliedto the tissue sections for 30 minutes each. Between antibodyapplications the sections were washed with TBS to remove unboundantibody. Sites of bound alkaline phosphatase were identified usingbrom-chloro-indolyl phosphate and nitro blue tetrazolium as the enzymesubstrate. After incubating the sections for 30 minutes at roomtemperature, the reaction was stopped by washing the sections in coldtap water. The slides were then air-dried and mounted in glycerinejelly. The sections were examined using bright field light microscopy inorder to establish the presence or absence of immunostaining, and itsdistribution.

SDS-PAGE and immunoblotting was followed by enhanced chemiluminescenece(ECL) technique as described below. Immunoblotting was also performedwith a monoclonal antibody to β-actin (clone no. AC-15, Sigma, Poole,Dorset, UK) to show the presence of a positive control protein in tumourand normal samples and indicate that there was no evidence of proteindegradation in any of the tissue samples.

The presence of CYP1B1 in each tumour that showed CYP1B1 byimmunohistochemistry, and the absence of detectable CYP1B1 in normaltissues, was confirmed by Western blot analysis. The proteins subjectedto Western blot analysis were from isolated microsome preparations.

Microsomes were prepared essentially as described (32). Tissue sampleswere thawed in 25 ml 0.01M Tris-HCl buffer, pH 7.4, containing 1.15% KClbefore being homogenized in 0.01 M Tris-HCl buffer, pH 7.4, containing0.25 M sucrose, 15% glycerol, using an Ultra-Turrax homogenizer (type TP18/2; Janke and Kunkel AG, Staufen Breisgau, Germany). Aftercentrifugation at 15,000×g for 20 min, the supernatant was removed andrecentrifuged at 116,000×g for 50 min. The pellet was resuspended afirst time in 0.1M Tris-HCl buffer, pH 7.4, containing 15% glycerol, 1mM EDTA, and recentrifuged at 116,000×g for 50 min. The pellet wasresuspended a second time in Tris-HCl-glycerol-EDTA buffer. Microsomalprotein concentration was determined (34).

A discontinuous polyacrylamide gel system, as described (33) withmodifications (32), was employed for separation of proteins in themicrosomes. 20 μl of a 1 mg/ml preparation of normal samples, and 40 μlof a 0.5 mg/ml preparation of tumour samples, in 0.125M Tris-HCl, pH6.8, containing 2.35% (w/v) sodium dodecyl sulfate, 5% (v/v)2-mercaptoethanol, and 0.005% bromophenol blue tracking dye were loadedonto the gel. 10 μl of a 1 mg/ml preparation of human liver microsomeswere used as positive controls. Samples were run on a 10% non-gradientgel at 30 mA.

Following SDS-PAGE, resolved proteins were blotted onto a nitrocellulosemembrane (Schleicher & Schuell; Dassel, Germany) overnight as described(35). Nonspecific binding sites were blocked with PBS containing 2%(w/v) nonfat milk, 0.05% (v/v) TWEEN 20™ for 30 minutes at roomtemperature, with continuous shaking. This buffer was also used forwashing stages. The nitrocellulose membrane was then incubated withCYP1B1-specific antibody (1:1000) for 90 minutes and goat anti-rabbitimmunoglobulin horseradish peroxidase conjugate (1:2000 Bio-RadLaboratories, Hemel Hempstead, Herts, UK) for 60 minutes. The membranewas washed for three successive 15-minute periods and one 60-minuteperiod after each incubation to remove unbound antibody. Boundhorseradish peroxidase was then visualized with an EnhancedChemiluminescence (ECL) kit (Amersham International, Aylesbury, Bucks,UK). Detection was carried out as described in the ECL protocol, withthe X-ray film (Hyperfilm-ECL; Amersham) being exposed for 30 seconds.

EXAMPLE 1

Expression of CYP1B1 in normal kidney and kidney tumours wereinvestigated.

Nephrectomy specimens (n=10) excised from primary renal cell carcinomawere used. Samples or normal kidney were taken at least severalcentimetres distant from the edge of each tumour, and onlymacroscopically viable tumour was sampled. Normal human livers (n=5)were obtained from renal transplant donors and stored at −80° C. priorto use.

Microsomes of normal kidney, kidney tumours and normal liver wereprepared and subjected to the SDS-PAGE and immunoblotting procedures inan enhanced chemiluminescence technique (21, 22) using an anti-CYP1polyclonal antibody. Recognition of human CYP1B1 was demonstrated usinga maltose-binding recombinant CYP1B1 fusion protein expressed in E.coli. Expressed CYP1A1 and CYP1A2 were supplied by Dr C L Crespi,Gentest Corp, Mass., USA. The results are shown in FIG. 1: lane 1 humanliver, lane 2 expressed recombinant CYP1B1 protein lanes 3, 5, 7, 9, 11normal kidney samples, lanes 4, 6, 8, 10, 12 corresponding kidneytumours. The same amount of microsomal protein (30 μg) was loaded intoeach lane, thus allowing direct comparison between the kidney and liversamples.

As shown in FIG. 1, the kidney tumours and expressed CYP1B1 show asingle immunoreactive band at 60 kDa corresponding to the molecularweight of expressed CYP1B1. In normal kidney none of the samples showedan immunoreactive band at 60 kDa. In addition, none of the kidneytumours or normal kidney samples showed the presence of CYP1A1.

Immunoblotting of liver samples an immunoreactive band at 54 kDacorresponding to the molecular weight of CYP1A2. The intensity of theband at 54 kDa showed liver-to-liver variation, whereas there was noCYP1B1 immunoreactive band at 60 kDa in any of the liver samples.

EXAMPLE 2

The expression of CYP1B1 was also investigated in breast cancer usingimmunoblotting.

Samples of breast tissue were obtained from patients undergoing surgeryeither for primary breast cancer or non-neoplastic breast disease.Immunoblotting was performed on breast cancers obtained from sixpatients (age range 45-67; three non-smokers, information not availablefor three patients), and histologically all these tumours werecarcinomas of no special type. The tissue samples were frozen in liquidnitrogen and stored at −80° C. prior to analysis.

SDS-PAGE and immunoblotting were carried out as described previously.CYP1B1 was detected using the anti-CYP1 polyclonal antibody referred toabove. The results are shown in FIG. 2; lane 1 human liver, lane 2expressed CYP1B1, lanes 3-8 breast tumors. As can be seen, a singleprotein band of molecular weight 60 kDa corresponding to the molecularweight of the expressed CYP1B1 protein was identified. As previously,CYP1B1 was not detectable in the liver sample, but CYP1A2 was detected.

EXAMPLE 3

Immunohistochemistry was used to demonstrate the presence of CYP1B1specifically in a variety of normal and tumour tissues. The results areshown in Table 1 and in FIG. 3.

Immunohistological localisation of CYP1B1 was investigated in tumoursand normal tissues from invasive ductal carcinoma of the breast,endometrial adenocarcinoma, transitional cell carcinoma of the bladder,diffuse high grade malignant lymphoma, high grade astrocytoma of thebrain, soft tissue sarcoma (malignant fibrous histocytoma), normalliver, normal kidney, normal small intestine. The antibody used was the218A anti-CYP1B1 polyclonal antibody described above.

FIG. 3 shows an immunoblot of CYP1B1 in different types of tumours andnormal tissues. Lane 1 normal colon, lane 2 colon adenocarcinoma, lane 3normal kidney, lane 4 carcinoma of kidney, lane 5 normal breast, lane 6breast cancer, lane 7 normal jejunum, lane 8 normal stomach, lane 9normal liver, lane 10 malignant mixed Müllerian tumour, lane 11endometrial adenocarcinoma, lane 12 ovarian carcinoma, lane 13 diffuse Bcell lymphoma, lane 14 transitional cell carcinoma, lane 15 lungcarcinoma, lane 16 positive control (dioxin-induced ACHN kidney tumourcells (panel A only). In panel B, the same series of tissue samples havebeen immunoblotted for β-actin, which is present in all normal andtumour samples. A Coomassie blue stained polyacrylamide gel of the sameseries of tissue samples displayed no evidence of protein degradation.The results demonstrated that this P450 is specifically localised totumour cells, and that there is no CYP1B1 immunoreactivty in normaltissues.

TABLE 1 CYP1B1 Expression in Tumour Tissues and Normal Tissues. NormalTumor no pos./ no pos./ Tissue no tested no tested Histopathologicaldiagnosis Bladder 0/8 8/8 transitional cell carcinoma Brain  0/12 11/12astrocytoma Breast  0/10 12/12 invasive ductal carcinoma Colon  0/1011/12 adenocarcinoma Connective 0/9 8/9 sarcoma tissue Kidney  0/1111/11 clear cell carcinoma n = 10; transitional cell carcinoma n = 1Liver 0/8 not tested not tested Lung 0/8 7/8 squamous carcinoma Lymphnode 0/5 9/9 non-Hodgkin's lymphoma Oesophagus 0/8 8/8 squamouscarcinoma Ovary not tested 7/7 adenocarcinoma Skin 0/6 6/6 squamouscarcinoma Small 0/5 not tested not tested Intestine Stomach  0/10  9/10adenocarcinoma Testis 0/8 14/14 malignant germ cell tumours Uterus 0/57/7 adenocarcinoma n = 5; malignant mixed Mullerian tumour n = 2 Total 0/123 128/133

EXAMPLE 4

Experiments were conducted to defect CYP1B1 RNA in various tumour andnormal tissues.

Reverse transcription polymerase chain reaction (RT-PCR) experiments todetect CYP1B1 mRNA were carried out as described in McKay et al (23).RNA was extracted from tissue samples and cDNA was synthesised from theisolated RNA using oligo (dT). The CYP1B1 primers had the followingsequences: Forward 5′-AAC TCT CCA TCA GGT GAG GT-3′ (nt 2104-2123);Reverse 5′-TAA GGA AGT ATA CCA GAA GGC-3′ (nt 2573-3593) giving a PCRproduct of 489 bp. β-actin was used as a positive control to confirm thepresence and integrity of mRNA in each sample and the β-actin primerswhich were brought from Stratagene (Cambridge, UK) had the followingsequences: Forward 5′-TGA CGG GGT CAC CCA CAC TGT GCC CAT CTA-3′ (nt1067-1105); Reverse 5′-CTA GAA GCA TTT GCG GTG GAC GAT GGA GGG-3′ (nt1876-1905). PCR with 35 cycles of amplification for both CYP1B1 andβ-actin was performed as described (23). The positive control for CYP1B1was a 2.78 kb CYP1B1 cDNA and the negative control was sterile water inplace of cDNA. After PCR 10 μl of the PCR product was electrophoresed ona 1.5% agarose gel which incorporated 0.007% w/v ethidium bromide andvisualized by UV illumination. The CYP1B1 PCR product was sequenced,after purification, by the direct dideoxy sequencing technique with a T7sequencing kit (Pharmacia, Milton Keynes, UK) used according to themanufacturer's protocol. To further investigate the relative amount ofCYP1B1 mRNA in normal and tumour tissues, semi-quantitative RT-PCR ofnormal and tumour kidney samples was performed using serial dilution ofcDNA (24). β-actin mRNA was used as an internal control (29).

FIG. 4 shows CYP1B1 and β-actin mRNA in normal (A and B) andcorresponding tumour (C and D) samples which have been detected byRT-PCR. Lane 1 kidney, lane 2 colon, lane 3 skin, lane 4 oesophagus,lane 5 stomach, lane 6 lymph node, lane 7 breast.

Analysis of the tumours by RT-PCR showed that all tumour samples inwhich CYP1B1 had been identified contained CYP1B1 mRNA. The PCR productwas of the expected molecular size when analyzed by agarose geleletrophoresis. Sequencing of the PCR product confirmed identity withCYP1B1.

Concluding remarks

The absence or low level of individual forms of P450 in most studies ofhuman cancer (15-18), combined with extrapolation from studies of rodenthepatic carcinogenesis (25), had led to the general belief that tumourcells do not significantly express P450. However, we have now shown thatCYP1B1 is expressed in a wide variety of malignant tumours of differenthistogenetic types and is not present in normal tissues, indicating thatthis P450 is a tumour specific form of P450. Tumours are composed of avariable proportion of tumour cells and non-tumour cells. To identifythat a protein is tumour specific, it is important to demonstrate thatthe protein is localised only to tumour cells. Immunohistochemistryallows the direct visualisation of tumour cells and has the spatialresolution to separate tumour cells from non-tumour cells. Furthermore,it is important to show there has been no differential degradation ofproteins in normal tissue samples compared with tumour samples andimmunoblotting for β-actin (as a positive control protein) showed it tobe present in every normal and tumour sample indicating there was noprotein degradation. In addition, Coomassie blue staining of thepolyacrylamide gels showed no evidence of protein degradation. Moreover,immunohistochemistry of the tumour samples provides its own internalcontrol as sections of tumour contain non-tumour cells.

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SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 4 <210> SEQ ID NO: 1 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE:1 aactctccat caggtgaggt 20 <210> SEQ ID NO: 2 <211> LENGTH: 21 <212>TYPE: DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 2 taaggaagtataccagaagg c 21 <210> SEQ ID NO: 3 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Homo sapiens <400> SEQUENCE: 3 tgacggggtc acccacactgtgcccatcta 30 <210> SEQ ID NO: 4 <211> LENGTH: 30 <212> TYPE: DNA <213>ORGANISM: Homo sapiens <400> SEQUENCE: 4 ctagaagcat ttgcggtggacgatggaggg 30

What is claimed is:
 1. A method for determining the presence of cancercells in a sample of tissue or cells from a human patient, the methodcomprising contacting CYP1B1 protein from said sample of said patientwith an antibody for human cytochrome P450 CYP1B1, and detecting bindingof the antibody to human CYP1B1 protein in said sample, wherein agreater amount of the binding of said antibody to said human CYP1B1protein in said sample as compared to normal control cells is anindication of the presence of cancer cells in said sample.
 2. The methodof claim 1, further comprising the steps of obtaining from the patientthe tissue sample to be tested for the presence of cancer cells; andproducing a prepared sample in a sample preparation process prior tocontacting the prepared sample with a CYP1B1 antibody.
 3. The method ofclaim 2 wherein binding of the antibody to CYP1B1 protein in sample isdetected by immunohistochemical analysis.
 4. The method of claim 3,wherein the sample preparation process comprises contacting the tissuewith a fixative and producing a thin section suitable forimmunohistochemical analysis.
 5. The method of claim 4, wherein thefixative is formalin.
 6. The method of claim 4, wherein the thin sectionis wax-embedded.
 7. The method of claim 3, wherein theimmunohistochemical analysis comprises a conjugated enzyme labellingtechnique.
 8. The method of claim 2, wherein the sample preparationprocess comprises tissue homogenization.
 9. The method of claim 8,wherein the sample preparation process further comprises isolatingmicrosomes.
 10. The method of claim 8, wherein the binding of theantibody to the CYP1B1 protein in a prepared sample is detected byWestern blot analysis.
 11. The method of claim 10, wherein the Westernblot analysis comprises a conjugated enzyme labelling technique.
 12. Themethod of claim 8, wherein the binding of the antibody to the CYP1B1protein in the prepared sample is detected by an immunoassay.
 13. Themethod of claim 12, wherein the immunoassay is selected from the groupconsisting of antibody capture assay, two-antibody sandwich assay andantigen capture assay.
 14. The method of claim 12, wherein theimmunoassay is a solid support-based immunoassay.
 15. The method ofclaim 12, wherein the immunoassay comprises a conjugated enzymelabelling technique.
 16. The method of claim 1, wherein the antibody isa polyclonal antibody.
 17. The method of claim 1, wherein the antibodyis a monoclonal antibody.
 18. The method of claim 1, wherein theantibody recognizes a preselected epitope of the CYP1B1 protein.
 19. Themethod of claim 1, wherein the antibody is specific for CYP1B1 protein.20. The method of claim 1, wherein the tissue sample is selected fromthe group consisting of bladder, brain, breast, colon, connectivetissue, kidney, lung, lymph node, oesophagus, ovary, skin, stomach,testis, and uterus.